Effects of a Neonicotinoid Insecticide and Population Density on Behavior and Development of Wood Frogs (Rana sylvatica). Submitted to Environmental Toxicology and Chemistry

Jeremie Bouffard, Department of Biology, University of Ottawa
Vincent Careau, Department of Biology, University of Ottawa
Stacey Robinson, Ecotoxicology and Wildlife Health Division, Wildlife and Landscape Science Directorate, Science and Technology Branch, Environment and Climate Change Canada
Patrick Bergeron*, Department of biological sciences, Bishop's University
*Corresponding author: pbergero@ubishops.ca

Goal: Using mesocosms, our goal was to determine the effects of pulse exposure to a neonicotinoid (imidacloprid) in the presence of a natural stressor (population density) on larval wood frog behaviors, development, and survival across metamorphosis.

Patrick Bergeron is responsible for managing the data and writing the code.


List of file names:


MSOF.csv

Main file for behavioral measurements taken during open field tests.

ID_Test: A combination of the tadpole ID and the test, separated by "_"
ID: Identification of the tadpole/froglet based on treatment (A, B, C, or D), Replicate tank number for each treatment (1, 2, or 3) and tadpole ID within a tank (A to Y, 25 per tank)
Test: Behavioral test performed (1, 2, or 3) based on Gosner stage (Gs; ~30, 42, or 45/46 respectively)
Phase: Whether the predator cue is absent (1) or present (2) during the behavioral test. Absence (1) always precedes presence (2) which only applies to Test 1 and 2 (Gs ~30, 42)
Arena: Arena in which the tadpole was filmed (Arena 1, 2, 3, 4, or NA if Test 3 (Gs 45/46, terrestrial arena). Arenas were always in the exact same position and orientation
Tank: Combination of treatment (A, B, C, or D) and replicate tank number (1, 2, or 3)
Treatment: Treatment the tadpole was exposed to which is a combination of density (low/high) and imidacloprid (neonicotinoid; absent/present) labelled as:
	A) Low/Absent
	B) High/Absent
	C) Low/Present
	D) High/Present
Density: Population density treatment within a tank (low or high; 0.33 tadpole/L or high: 1 tadpole/L respectively)
IMI: Neonicotinoid  (Imidacloprid) treatment (No or Yes; absent or present respectively)
Date: Date the tadpole/froglet underwent the behavioral test (YYYY-MM-DD)
Jdate: "Julian" date from May 1st of the test (conversion of the date to a simplified numerical format as days since May 1st 2019)
Time: Time of day the tadpole/froglet underwent the behavioral test
Acc: Time spent acclimating by the tadpole prior to a behavioral test (in minutes). Test 3 tadpoles did not have measured acclimation time
Temp: Temperature of the water/air within each arena during a given aquatic or terrestrial behavioral test (degrees Celsius)
TIME: Recoding of the Time variable to a 24h clock to calculate TIME_Min.
TIME_Min: Time of day of the test converted to minutes from midnight (i.e. 10:53am = 653)
Dist: Distance travelled by the tadpole in the open field arena during the specific Test/Phase (pixels converted to cm)
Moving: Time spent moving in seconds
AvgSp: Average speed of the tadpole calculated as distance travelled divided by time spent moving (cm/s)
Latency: Latency to first movement (time spent not moving at the start of the experiment) in seconds. Max 120 seconds
Center: Time spent in the center of the arena (64% of the arena's surface area) measured in seconds (Based on the tadpoles own arena; Center_1, Center_2, Center_3, or Center_4). Max 120 seconds
Salvage: Light quality within the open field box (Light (normal), dark, NA). For some tests, the power of the light decreased during the experiment due to diminishing battery life leading to the dark category (the lighting was still present but weaker). These were not significantly different from normal light condition tests.
ETHO_TRIAL: Order in which the test was uploaded/treated within EthoVision (for tracking purposes in case tracking issues were noticed)
Center_1: Time spent in the center of arena 1 (0 if the tadpole was not in that arena during the behavioral test)
Center_2: Time spent in the center of arena 2 (0 if the tadpole was not in that arena during the behavioral test)
Center_3: Time spent in the center of arena 3 (0 if the tadpole was not in that arena during the behavioral test)
Center_4: Time spent in the center of arena 4 (0 if the tadpole was not in that arena during the behavioral test)
Air.Temp.: Temperature of the air during terrestrial tests (in degrees Celsius)
Weather: Weather conditions during behavioral tests (Sunny/Cloudy) based on visually assessed cloud cover
Center80: Time spent in the center of the arena if the center made up 80% of the arena instead of 64% (did not change the results significantly)
LeftZone: Time spent in the left side of the terrestrial (Test 3, Gs 45/46) arena
RightZone: Time spent in the right side of the terrestrial (Test 3, Gs 45/56) arena
Floor: Time spent on the floor of the terrestrial (Test 3, Gs 45/56) arena
Walls: Time spent on the wall of the terrestrial (Test 3, Gs 45/56) arena
Wall_Blind: The direction in which a wall or blind was present during the terrestrial behavioral test in order to provide uniform ambient lighting (prevent sunlight from entering the arena creating unstanderdized lighting/shading conditions as the sun moved across the sky or was hidden behind clouds (Left, Right, or No if the wall/blind was not needed at that time of day)
zDist: Square rooted and scaled distance travelled
zMov: Square rooted and scaled time spent moving
zAvsp: Square rooted and scaled average speed
zLat: Square rooted and scaled Latency to first movement
zCen: Square rooted and scaled time spent in the center of the arena
zDens: Density treatment where low density = -0.5, and high density = 0.5
zIMI: Imidacloprid treatment where absent (no imidacloprid) = -0.5, and present (imidacloprid present) = 0.5
zPha: Phase (i.e. absence and presence of predation cue during aquatic behavioral tests) recoded as -0.5 for absences of cue, and 0.5 for presence of cue
ID_Phase: Combination of tadpole ID and Phase (presence/absence of predation cue within aquatic tests; 1 or 2) separated by "_"
ID_Test_Phase: Combination of tadpole ID, Test (1, 2, or 3; Gs ~30, 42, 45/46 respectively) and Phase (presence/absence of predation cue within aquatic tests; 1 or 2 (only 1 is available for Test 3, the terrestrial test)) separated by "_"
ASA: Average surface area calculated as the average of 10 dorsal surface area measurements made in ImageJ on the same image of a given tadpole (to control for human error).
Weight: Weight in grams of the froglets after euthanasia
EmergenceDate: Date each froglet reached Gosner stage 45/46 and underwent the third behavioral test


AllMasses.csv

Emergence date and mass data for cupped (focal individuals) and free swimming tadpoles.

ID: Identification of the tadpole/froglet based on treatment (A, B, C, or D), replicate tank number for each treatment (1, 2, or 3) and tadpole ID within a tank (A to Y for Identified/focal individuals, 25 per tank; FS for free swimmers with following numbers being weighing order)
Tank: Combination of treatment (A, B, C, or D) and replicate tank number (1, 2, or 3)
Cupped: Whether the froglet was in a cup or a free swimmer within the tank (Yes or No; cupped or free swimmer)
Treatment: Treatment the tadpole was exposed to which is a combination of density (low/high) and imidacloprid (neonicotinoid; absent/present) labelled as:
	A) Low/Absent
	B) High/Absent
	C) Low/Present
	D) High/Present
Density: Population density treatment within a tank (low or high; 0.33 tadpole/L or high: 1 tadpole/L respectively)
IMI: Neonicotinoid  (Imidacloprid) treatment (No or Yes; absent or present respectively)
Jdate: "Julian" date from May 1st of the test (conversion of the date to a simplified numerical format as days since May 1st 2019)
Weight: Froglet weight in grams after euthanasia
Date: Date the froglet reached stage 45/46

The following is the list of free swimmers that did not complete metamorphosis or were found as desiccated froglets by the end of the experiment (removed from mass analyses):
A2FS33, A2FS34, A2FS35, A3FS23, A3FS24, A3FS25, A3FS26, B1FS100, B1FS101, B1FS102, B1FS103, B1FS104, B1FS105, B1FS106, B1FS107, B1FS108, B1FS109, B1FS110, B1FS111, B1FS112, B1FS113, B2FS87, B2FS88, B2FS89, B2FS90, B2FS91, B2FS92, B2FS93, B2FS94, B2FS95, B2FS96, B2FS97, B2FS98, B2FS99, B2FS100, B2FS101, B2FS102, B2FS103, B2FS104, B2FS105, B2FS106, B2FS107, B2FS108, B2FS109, B2FS110, B2FS111, B2FS112, B2FS113, B2FS114, B2FS115, B3FS119, B3FS120, B3FS121, B3FS122, B3FS123, B3FS124, B3FS125, B3FS126, B3FS127, B3FS128, B3FS129, B3FS130, B3FS131, B3FS132, B3FS133, B3FS134, B3FS135, B3FS136, B3FS137, B3FS138, C1FS34, C3FS29, D1FS119, D1FS120, D1FS121, D1FS122, D2FS99, D2FS100, D2FS101, D2FS102, D2FS103, D2FS104, D2FS105, D2FS106, D2FS107, D2FS108, D2FS109, D2FS110, D2FS111, D2FS112, D2FS113, D2FS114, D3FS119, D3FS120, D3FS121, D3FS122, D3FS123, D3FS124, D3FS125


Temperatures.csv

These are the mesocosm water sample (for chemical analyses) metrics and temperatures throughout the experiment.

Date: Date the tank was sampled and/or water temperature of the mesocosm was measured
Tank: Combination of treatment (A, B, C, or D) and replicate tank number (1, 2, or 3)
Treatment: Treatment the tadpole was exposed to which is a combination of density (low/high) and imidacloprid (neonicotinoid; absent/present) labelled as:
	A) Low/Absent
	B) High/Absent
	C) Low/Present
	D) High/Present
Density: Population density treatment within a tank (low or high; 0.33 tadpole/L or high: 1 tadpole/L respectively)
Imi: Neonicotinoid  (Imidacloprid) treatment (No or Yes; absent or present respectively)
Temp: Temperature of the tank in degrees Celsius
pHtemp: Temperature of the sample when measuring pH
pH: pH of the tank sample
IMIppb: Imidacloprid concentration in parts per billion


SurvData.csv

Survival percentages of wood frogs at the end of the experiment.

Treat: Treatment the tadpole was exposed to which is a combination of density (low/high) and imidacloprid (neonicotinoid; absent/present) labelled as:
	A) Low/Absent
	B) High/Absent
	C) Low/Present
	D) High/Present
DensS: Population density treatment within a tank (low or high; 0.33 tadpole/L or high: 1 tadpole/L respectively)
ImiS: Neonicotinoid  (Imidacloprid) treatment (No or Yes; absent or present respectively)
SurvCupped: Percentage of cupped individuals in each mesocosm tank (treatment replicate) that survived prior to metamorphic climax (before some escaped their cups into the tanks and could not be differentiated from free swimmers)
SurvTotal: Percentage of individuals (total; cupped and free swimming) that survived to the end of the experiment in each mesocosm tank, including individuals that did not complete metamorphosis by the end of the experiment
SurvNoMetaMorph: Percentage of individuals (total; cupped and free swimming) that survived to the end of the experiment in each mesocosm tank, excluding individuals that did not complete metamorphosis by the end of the experiment


SR01-2019_Robinson-JBNEO2019-WaterSamples.xlsx

File tracking water samples and their imidacloprid concentrations.

Specimen no.: Identification of the sample taken.
Matrix: Medium in which chemical is found.
Type of Container (Volume): Type of container used to sample and store the water from the mesocosms or environment 
Number of samples: How many samples were in each bottle
Description: Description of where the sample came from.
Chemical being Evaluated (CAS): The chemical for which the concentration was determined
Treatment (Nominal; ug/L): Nominal concentration of the sample
Tadpole Density: Population density treatment within a tank (low or high; 0.33 tadpole/L or high: 1 tadpole/L respectively). NA was for when samples were taken from the environment
Collection Date: Date the samples were collected
Collection Time: Time of day the samples were taken
Collected Before/After H20 Change: Whether the samples were taken before or after imidacloprid was added to the tanks (does not apply for samples taken from the environment)
Pooled / Not pooled: Whether the sample originated from a single mesocosm (not pooled) or was the result of combining samples from replicate tanks (pooled; i.e. combining samples for a given treatment; used to assess degradation of imidacloprid) 
Specimen Source: Where the sample was taken (see "Description" for more details)
Collection Technique: Protocol used to sample the water
Collection Condition: Condition of the water at the time of sampling (fresh, old, etc.)
Storage upon Collection: The condition under which the samples were stored
Storage Location: Where the samples were stored within an institution
Concentration (ppb) / ND: Not Detected: Measured imidacloprid concentration of the samples
Method Detection Limit: Detection limits of the chemical analyses for the chemical of interest
Analyzed Date: Date the sample was analyzed
Batch: Grouping of samples which underwent chemical analyses together.
Analyst: Initials of the person who conducted the chemical analyses.


In all data sheets, n/a, na, and NA indicate missing values or values that were not applicaple.
All statistical analyses were conducted in R 3.6.3 (2020-02-29).
Platform: x86_64-w64-mingw32
Running under: Microsoft Windows 10 Home, Build 19043